CN102068698A - Nanometer vaccine and preparation method thereof - Google Patents

Abstract

The invention provides a nanometer vaccine and a preparation method thereof. A new mannose glycosylated-cationic liposome complex can be used as a vaccine vector and can be applied to the vaccine. In the new liposome complex, neutral phospholipid and mannose are mixed into cationic lipid, so the immunologic adjuvant effect and the antigen-presenting cell targeting property of a liposome are improved, and the cytotoxic effect of the liposome is reduced obviously. The invention provides a novel high-efficiency vaccine vector and an adjuvant which do not have side effects and can improve the immunologic effect of the vaccine obviously.

Description

Translated fromChinese

纳米疫苗及其制备方法Nano vaccine and preparation method thereof

【技术领域】【Technical field】

本发明涉及一种疫苗及其制备方法，特别涉及一种采用脂质体作为载体的疫苗及其制备方法。The invention relates to a vaccine and a preparation method thereof, in particular to a vaccine using liposome as a carrier and a preparation method thereof.

本发明又涉及一种疫苗载体。The present invention also relates to a vaccine carrier.

本发明还涉及一种可应用于疫苗载体的脂质体复合物。The invention also relates to a liposome complex applicable to vaccine carriers.

【背景技术】【Background technique】

疫苗是预防和控制传染性疾病发生及发展的重要手段。免疫佐剂(又称非特异性免疫增生剂)则是疫苗中不可或缺的组成成分，能够有效地促进疫苗诱导的特异性免疫反应。随着疫苗研究的飞速发展，新型的基因工程疫苗正被广泛用于疫苗的研制开发。这种疫苗具有纯度高、特异性强等优点，但是由于其分子小，免疫原性相对较差，迫切需要结合有效的免疫佐剂，以提高疫苗的免疫效力。过去的数十年来，人们在研究中发现和研制了各种新型的免疫佐剂，然而潜在的安全性及稳定性等因素大大制约了免疫佐剂的推广使用。Vaccines are an important means to prevent and control the occurrence and development of infectious diseases. Immune adjuvants (also known as non-specific immune proliferators) are indispensable components in vaccines, which can effectively promote specific immune responses induced by vaccines. With the rapid development of vaccine research, new genetically engineered vaccines are being widely used in the research and development of vaccines. This vaccine has the advantages of high purity and strong specificity, but due to its small molecule and relatively poor immunogenicity, it is urgent to combine effective immune adjuvants to improve the immune efficacy of the vaccine. Over the past few decades, people have discovered and developed various new immune adjuvants in research, but factors such as potential safety and stability have greatly restricted the promotion and use of immune adjuvants.

脂质体纳米颗粒是由磷脂双层壳包裹水相核心形成的球形的实体，其结构类似生物膜，是一种生物相容性好且无毒的纳米材料。它可包封水溶性和脂溶性药物，具有减少药物剂量、缓释、及靶向性释放药物等优点，因而被广泛用于纳米抗肿瘤药物的开发。此外，纳米脂质体也是一种优良的抗原载体，不仅能够包裹一系列理化性质不同的抗原及免疫佐剂，保护蛋白多肽抗原不被降解，还可以促进抗原呈递细胞对抗原的吞噬及呈递，提高机体的特异性免疫反应。基于以上这些优点，脂质体纳米颗粒作为一种新型的疫苗载体，正逐渐用于细菌疫苗、病毒疫苗、抗寄生虫疫苗以及抗肿瘤疫苗等研制开发。Liposome nanoparticles are spherical entities formed by phospholipid bilayer shells enclosing the aqueous phase core. Its structure is similar to biological membranes, and it is a kind of nanomaterials with good biocompatibility and non-toxicity. It can encapsulate water-soluble and fat-soluble drugs, and has the advantages of reduced drug dose, sustained release, and targeted release of drugs, so it is widely used in the development of nano anti-tumor drugs. In addition, nanoliposomes are also an excellent antigen carrier, which can not only encapsulate a series of antigens and immune adjuvants with different physical and chemical properties, protect protein and polypeptide antigens from degradation, but also promote the phagocytosis and presentation of antigens by antigen-presenting cells. Improve the body's specific immune response. Based on the above advantages, liposome nanoparticles, as a new type of vaccine carrier, are gradually being used in the research and development of bacterial vaccines, viral vaccines, anti-parasitic vaccines and anti-tumor vaccines.

目前最常用的脂质体蛋白疫苗载体是以中性磷脂和胆固醇为主要成分。这类中性脂质体纳米颗粒虽是一种优良的蛋白载体，却并不能直接促进免疫反应。此外，未经修饰的脂质体靶向性差，不能有效地被抗原呈递细胞(如树突状细胞及单核巨噬细胞)摄取。The most commonly used liposomal protein vaccine carrier is mainly composed of neutral phospholipids and cholesterol. Although such neutral liposomal nanoparticles are an excellent protein carrier, they cannot directly promote immune responses. In addition, unmodified liposomes have poor targeting and cannot be effectively taken up by antigen-presenting cells such as dendritic cells and monocyte-macrophages.

现有的脂质体疫苗多采用中性脂质体为载体，然而由于中性脂质体本身不能直接促进免疫反应且靶向性差，因此制约了疫苗的免疫效率。采用阳离子脂质取代中性磷脂虽能够促进免疫反应和抗原呈递细胞靶向性，过高的表面电荷却使其具有明显的细胞毒性。Most of the existing liposome vaccines use neutral liposomes as carriers. However, the neutral liposomes themselves cannot directly promote the immune response and have poor targeting, thus restricting the immune efficiency of the vaccine. Although the use of cationic lipids to replace neutral phospholipids can promote immune response and antigen-presenting cell targeting, the high surface charge makes them have obvious cytotoxicity.

综上所述，现有的脂质体应用于疫苗载体，有所缺失，难于满足需求。To sum up, the existing liposomes are used in vaccine carriers, but there are some deficiencies, and it is difficult to meet the demand.

【发明内容】【Content of invention】

有鉴于此，有必要针对现有脂质体疫苗载体的缺陷，提出一种新的甘露糖基化的阳离子脂质体复合物，可以作为疫苗载体及应用于疫苗，这种新的脂质体复合物在阳离子脂质中掺入中性磷脂和甘露糖，不但提高了脂质体的免疫佐剂作用和抗原呈递细胞靶向性，而且明显减低其细胞毒作用，提供了一种高效无副作用的新型疫苗载体和佐剂，能显著增强疫苗的免疫效果。In view of this, it is necessary to address the defects of existing liposome vaccine carriers, and propose a new mannosylated cationic liposome complex, which can be used as a vaccine carrier and applied to vaccines. This new liposome The complex incorporates neutral phospholipids and mannose in cationic lipids, which not only improves the immune adjuvant effect of liposomes and the targeting of antigen-presenting cells, but also significantly reduces its cytotoxicity, providing a highly effective and no side effects The new vaccine carrier and adjuvant can significantly enhance the immune effect of the vaccine.

为了实现上述的发明目的，本发明的技术方案如下：In order to realize above-mentioned purpose of the invention, technical scheme of the present invention is as follows:

本发明提供了一种甘露糖基化的阳离子脂质体复合物，包括按比例混合的阳离子脂、中性磷脂和甘露糖苷，阳离子脂和中性磷脂的摩尔比为19∶1至2∶18，甘露糖苷与包括阳离子脂和中性磷脂的脂质体的摩尔比为2∶98至20∶80。The invention provides a mannosylated cationic liposome complex, comprising cationic lipid, neutral phospholipid and mannoside mixed in proportion, the molar ratio of cationic lipid and neutral phospholipid is 19:1 to 2:18 , the molar ratio of mannoside to liposomes including cationic lipids and neutral phospholipids is 2:98 to 20:80.

优选地，阳离子脂是是带有正电荷两性分子，包括带有正电荷的头部和疏水尾部，带正电荷的极性头部含有氨基、季铵盐及多胺等胺类基团，而疏水尾部包括饱和或不饱和脂肪酸链或胆固醇环。Preferably, the cationic lipid is a positively charged amphoteric molecule, including a positively charged head and a hydrophobic tail, and the positively charged polar head contains amino groups such as amino groups, quaternary ammonium salts, and polyamines, while The hydrophobic tail includes saturated or unsaturated fatty acid chains or cholesterol rings.

优选地，阳离子脂包括双十烷基二甲基溴化铵(Didecyldimethylammoniumbromide，简称DDAB)、二油酰三甲基铵丙烷(ioleoyltrimethylammoniumpropane，简称DOTAP)、二油酰丙基氯化三甲铵(dioleoylpropyltrimethylammonium，简称DOTMA)、二甲氨基乙基氨甲酰基-胆固醇(3-(N-(N′，N′-Dimethylaminoethane)carbamoyl)cholesterol，简称DC-Chol)和二油酰磷脂酰胆碱醚(dioleyl ether phosphatidylcholine，简称DOEPC)中的一种或多种的组合。Preferably, the cationic lipids include didecyldimethylammoniumbromide (DDAB for short), dioleoyltrimethylammonium propane (DOTAP for short), dioleoylpropyltrimethylammonium chloride (dioleoylpropyltrimethylammonium, DOTMA for short), dimethylaminoethyl carbamoyl-cholesterol (3-(N-(N',N'-Dimethylaminoethane) carbamoyl) cholesterol, DC-Chol for short) and dioleyl phosphatidylcholine ether (dioleyl ether Phosphatidylcholine, referred to as DOEPC) in one or more combinations.

优选地，中性磷脂是两性分子，包括磷酸相连的取代基团(含氨碱或醇类)构成的亲水头和脂肪酸链构成的疏水尾，表面静电荷基本接近0。Preferably, the neutral phospholipid is an amphiphilic molecule, including a hydrophilic head formed by phosphate-linked substituent groups (ammonia-containing bases or alcohols) and a hydrophobic tail formed by fatty acid chains, and the surface electrostatic charge is basically close to zero.

优选地，中性磷脂包括二油酰磷脂酰胆碱(Dioleoylphatidylcholine，简称DOPC)、二豆蔻酰磷脂酰胆碱(dimyristoylphosphatidylcholine，简称DMPC)、二亚油酰磷脂酰胆碱(dilinoleoylphosphatidylcholine，简称DLPC)、二棕榈酰磷脂酰胆碱(Dipalmitoylphosphatidylcholine，简称DPPC)、二硬脂酰磷脂酰胆碱(distearoylphosphatidylcholine，简称DSPC)、二肉豆蔻酰磷酸乙醇胺(Dimyristoylphosphatidylethanolamine，DMPE)、二棕榈酰磷脂酰乙醇胺(Dipalmitoylphosphatidylethanolamine，简称DPPE)和二硬脂酰磷脂酰乙醇胺(distearoylphosphatidylethanolamine，简称DSPE)中的一种或多种的组合。Preferably, the neutral phospholipids include dioleoylphosphatidylcholine (DOPC for short), dimyristoylphosphatidylcholine (DMPC for short), dilinoleoylphosphatidylcholine (DLPC for short), Dipalmitoylphosphatidylcholine (DPPC for short), distearoylphosphatidylcholine (DSPC for short), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (Dipalmitoylphosphatidylethanolamine, DPPE for short) and a combination of one or more of distearoylphosphatidylethanolamine (DSPE for short).

优选地，甘露糖苷包括甲基-D-甘露糖苷(Methyl-α-D-mannopyranoside)、4-硝基苯基-α-D-吡喃甘露糖苷(4-Nitrophenyl-α-D-mannopyranoside)、4-甲基伞形酮基-α-D-吡喃甘露糖苷(4-Methylumbelliferyl-α-D-mannopyranoside)和4-氨基苯基-D-甘露糖苷(4-Aminophenylα-D-mannopyranoside)中的一种或多种的组合。Preferably, mannosides include methyl-D-mannoside (Methyl-α-D-mannopyranoside), 4-nitrophenyl-α-D-mannopyranoside (4-Nitrophenyl-α-D-mannopyranoside), In 4-methylumbelliferyl-α-D-mannopyranoside (4-Methylumbelliferyl-α-D-mannopyranoside) and 4-aminophenyl-D-mannoside (4-Aminophenylα-D-mannopyranoside) One or more combinations.

本发明又提供了一种具有免疫佐剂功效的疫苗载体，包裹疫苗抗原，疫苗载体为甘露糖基化的阳离子脂质体复合物，包括按比例混合的阳离子脂、中性磷脂和甘露糖苷，阳离子脂和中性磷脂的摩尔比为19∶1至2∶18，甘露糖苷与包括阳离子脂和中性磷脂的脂质体的摩尔比为2∶98至20∶80。The present invention also provides a vaccine carrier with immune adjuvant effect, which encapsulates the vaccine antigen. The vaccine carrier is a mannosylated cationic liposome complex, including cationic lipids, neutral phospholipids and mannosides mixed in proportion, The molar ratio of cationic lipid to neutral phospholipid is 19:1 to 2:18, and the molar ratio of mannoside to liposome including cationic lipid and neutral phospholipid is 2:98 to 20:80.

优选地，阳离子脂是是带有正电荷两性分子，包括带有正电荷的头部和疏水尾部，带正电荷的极性头部含有氨基、季铵盐及多胺等胺类基团，而疏水尾部包括饱和或不饱和脂肪酸链或胆固醇环。Preferably, the cationic lipid is a positively charged amphoteric molecule, including a positively charged head and a hydrophobic tail, and the positively charged polar head contains amino groups such as amino groups, quaternary ammonium salts, and polyamines, while The hydrophobic tail includes saturated or unsaturated fatty acid chains or cholesterol rings.

优选地，阳离子脂包括DDAB、DOTAP、DODAP、DOTMA和DOEPC中的一种或多种的组合。Preferably, the cationic lipid comprises one or a combination of DDAB, DOTAP, DODAP, DOTMA and DOEPC.

优选地，中性磷脂是两性分子，包括磷酸相连的取代基团(含氨碱或醇类)构成的亲水头和脂肪酸链构成的疏水尾，表面静电荷基本接近0。Preferably, the neutral phospholipid is an amphiphilic molecule, including a hydrophilic head formed by phosphate-linked substituent groups (ammonia-containing bases or alcohols) and a hydrophobic tail formed by fatty acid chains, and the surface electrostatic charge is basically close to zero.

优选地，中性磷脂包括DOPC、DMPC、DLPC、DPPC、DSPC、DMPE、DPPE和DSPE中的一种或多种的组合。Preferably, the neutral phospholipids include one or more of DOPC, DMPC, DLPC, DPPC, DSPC, DMPE, DPPE and DSPE in combination.

优选地，甘露糖苷包括甲基-D-甘露糖苷、4-硝基苯基-α-D-吡喃甘露糖苷、4-甲基伞形酮基-α-D-吡喃甘露糖苷和4-氨基苯基-D-甘露糖苷中的一种或多种的组合。Preferably, the mannosides include methyl-D-mannoside, 4-nitrophenyl-α-D-mannopyranoside, 4-methylumbelliferyl-α-D-mannopyranoside and 4- One or more combinations of aminophenyl-D-mannosides.

本发明再提供了一种纳米疫苗，包括疫苗抗原和包裹疫苗抗原的脂质体，脂质体采用甘露糖基化的阳离子脂质体复合物，包括按比例混合的阳离子脂、中性磷脂和甘露糖苷，阳离子脂和中性磷脂的摩尔比为19∶1至2∶18，甘露糖苷与包括阳离子脂和中性磷脂的脂质体的摩尔比为2∶98至20∶80。The present invention provides a kind of nano-vaccine again, comprises the liposome of vaccine antigen and encapsulation vaccine antigen, and liposome adopts the cationic liposome complex of mannosylation, comprises cationic lipid, neutral phospholipid and The molar ratio of mannoside, cationic lipid and neutral phospholipid is 19:1 to 2:18, and the molar ratio of mannoside to liposome including cationic lipid and neutral phospholipid is 2:98 to 20:80.

优选地，阳离子脂是是带有正电荷两性分子，包括带有正电荷的头部和疏水尾部，带正电荷的极性头部含有氨基、季铵盐及多胺等胺类基团，而疏水尾部包括饱和或不饱和脂肪酸链或胆固醇环。Preferably, the cationic lipid is a positively charged amphoteric molecule, including a positively charged head and a hydrophobic tail, and the positively charged polar head contains amino groups such as amino groups, quaternary ammonium salts, and polyamines, while The hydrophobic tail includes saturated or unsaturated fatty acid chains or cholesterol rings.

优选地，阳离子脂包括DDAB、DOTAP、DODAP、DOTMA和DOEPC中的一种或多种的组合。Preferably, the cationic lipid comprises one or a combination of DDAB, DOTAP, DODAP, DOTMA and DOEPC.

优选地，所述中性磷脂是两性分子，包括磷酸相连的取代基团(含氨碱或醇类)构成的亲水头和脂肪酸链构成的疏水尾，表面静电荷基本接近0。Preferably, the neutral phospholipid is an amphiphilic molecule, including a hydrophilic head composed of phosphoric acid-linked substituent groups (ammonia-containing bases or alcohols) and a hydrophobic tail composed of fatty acid chains, and the surface electrostatic charge is basically close to zero.

优选地，中性磷脂包括DOPC、DMPC、DLPC、DPPC、DSPC、DMPE、DPPE和DSPE中的一种或多种的组合。Preferably, the neutral phospholipids include one or more of DOPC, DMPC, DLPC, DPPC, DSPC, DMPE, DPPE and DSPE in combination.

优选地，甘露糖苷包括甲基-D-甘露糖苷、4-硝基苯基-α-D-吡喃甘露糖苷、4-甲基伞形酮基-α-D-吡喃甘露糖苷和4-氨基苯基-D-甘露糖苷中的一种或多种的组合。Preferably, the mannosides include methyl-D-mannoside, 4-nitrophenyl-α-D-mannopyranoside, 4-methylumbelliferyl-α-D-mannopyranoside and 4- One or more combinations of aminophenyl-D-mannosides.

优选地，抗原为各类蛋白、多肽、多糖、DNA或RNA，所述抗原来自于病毒、细菌、其他微生物、肿瘤或基因工程蛋白产物。Preferably, the antigen is various proteins, polypeptides, polysaccharides, DNA or RNA, and the antigens are derived from viruses, bacteria, other microorganisms, tumors or genetically engineered protein products.

优选地，疫苗的免疫方式为皮下注射或肌肉注射。Preferably, the immunization method of the vaccine is subcutaneous injection or intramuscular injection.

本发明还提供了一种具有免疫佐剂功效的疫苗载体的制造方法，包括如下步骤：取阳离子脂、中性磷脂和甘露糖苷分别溶于氯仿-甲醇，然后按比例混合后置于容器中；将容器中的混合物吹干成一层均匀的薄膜，而后进行真空干燥处理；加入含有抗原的缓冲液，然后进行水化处理及水浴超声处理，挤压过聚碳酸酯膜，冷藏放置备用。The present invention also provides a method for producing a vaccine carrier with immune adjuvant effect, comprising the following steps: dissolving cationic lipids, neutral phospholipids and mannoside in chloroform-methanol respectively, mixing them in proportion and placing them in a container; Dry the mixture in the container to form a uniform film, and then carry out vacuum drying treatment; add buffer solution containing antigen, then perform hydration treatment and water bath ultrasonic treatment, extrude the polycarbonate film, and refrigerate for future use.

优选地，氯仿-甲醇中氯仿和甲醇的体积比为2∶1。Preferably, the volume ratio of chloroform and methanol in chloroform-methanol is 2:1.

优选地，容器中的混合物用稳定的氮气流旋转吹干。Preferably, the mixture in the container is rotary blown dry with a steady stream of nitrogen.

优选地，真空干燥处理是在真空干燥箱中真空干燥过夜，第二天加入含有抗原的缓冲液。Preferably, the vacuum drying treatment is vacuum drying overnight in a vacuum drying oven, and adding the buffer solution containing the antigen the next day.

优选地，加入含有抗原的磷酸盐(Phosphate buffered saline，简称PBS)缓冲液后，放置于4℃水化12小时，水浴超声10分钟，挤压过聚碳酸酯膜两次，4℃放置备用。Preferably, after adding a phosphate buffered saline (PBS) buffer solution containing antigen, place it at 4°C for 12 hours of hydration, ultrasonicate in a water bath for 10 minutes, squeeze the polycarbonate membrane twice, and place it at 4°C for use.

由于采用上述的技术方案，本发明的有益效果如下：Owing to adopting above-mentioned technical scheme, the beneficial effects of the present invention are as follows:

本发明的甘露糖基化的阳离子脂质体复合物在阳离子脂中添加一定比例的中性磷脂和甘露糖，合成表面携带正电荷和甘露糖基的阳离子脂质体复合物，不但大大提高了脂质体靶向抗原呈递细胞的能力，而且显著增强了脂质体的免疫佐剂作用。与传统的中性脂质体相比，在功能上有了很大的改善，本发明的阳离子脂质体复合物不但具有强大的免疫佐剂功效，还能更加有效地促进免疫细胞对抗原的摄取呈递，促进疫苗的免疫效果。此外掺入一定比例中性磷脂成份还能明显减低阳离子脂的细胞毒作用，使疫苗更加安全。The mannosylated cationic liposome complex of the present invention adds a certain proportion of neutral phospholipids and mannose in the cationic lipid, and synthesizes the cationic liposome complex with positive charges and mannose groups on the surface, which not only greatly improves the The ability of liposomes to target antigen-presenting cells significantly enhanced the immune adjuvant effect of liposomes. Compared with traditional neutral liposomes, the function has been greatly improved. The cationic liposome complex of the present invention not only has a powerful immune adjuvant effect, but also can more effectively promote immune cells to antigens. Ingestion and presentation to promote the immune effect of the vaccine. In addition, the addition of a certain proportion of neutral phospholipids can also significantly reduce the cytotoxic effect of cationic lipids, making the vaccine safer.

本发明的甘露糖基化的阳离子脂质体复合物应用于疫苗载体，可以同时包裹一种或多种抗原，不仅可以促进抗原的靶向传递，而且显著提高抗原诱导的免疫反应，增强疫苗的免疫效力，且安全无毒副作用。The mannosylated cationic liposome complex of the present invention is applied to a vaccine carrier and can encapsulate one or more antigens at the same time, which can not only promote the targeted delivery of the antigen, but also significantly improve the immune response induced by the antigen, and enhance the efficacy of the vaccine. Immune effect, and safe and non-toxic side effects.

本发明的疫苗以一种新型的甘露糖基化阳离子脂质体复合物作为疫苗载体，提高疫苗的免疫效力，构建安全高效的纳米疫苗。The vaccine of the invention uses a novel mannosylated cationic liposome complex as a vaccine carrier, improves the immune efficacy of the vaccine, and constructs a safe and efficient nano-vaccine.

【附图说明】【Description of drawings】

为更进一步了解本发明的特征及技术内容，请参阅以下有关本发明的附图，然而所附图式仅提供参考与说明用，并非用来对本发明加以限制。In order to further understand the features and technical content of the present invention, please refer to the following drawings related to the present invention. However, the attached drawings are provided for reference and illustration only, and are not intended to limit the present invention.

图1是本发明的以甘露糖基化的阳离子脂质体复合物为载体的纳米疫苗的结构示意图；Fig. 1 is the structural representation of the nano-vaccine of the present invention with the cationic liposome complex of mannosylation as carrier;

图2是本发明的纳米疫苗的制备方法的流程图；Fig. 2 is the flowchart of the preparation method of nano vaccine of the present invention;

图3是本发明的阳离子脂质体复合物诱导单核细胞活化并表达CD86分子对比图；Fig. 3 is that the cationic liposome complex of the present invention induces monocyte activation and expresses CD86 molecule contrast figure;

图4是本发明的阳离子脂质体复合物诱导树突状细胞表达CD83对比图；Fig. 4 is a comparison chart of dendritic cells expressing CD83 induced by the cationic liposome complex of the present invention;

图5是本发明的阳离子脂质体复合物促进巨噬细胞对抗原(BSA-FITC)的摄取对比图；Fig. 5 is that cationic liposome complex of the present invention promotes macrophage to absorb the contrast figure of antigen (BSA-FITC);

图6是阳离子脂质体复合物对单核细胞存活率的影响对比图。Figure 6 is a comparison chart of the effect of cationic liposome complexes on the survival rate of monocytes.

【具体实施方式】【Detailed ways】

下面结合附图，通过对本发明的具体实施方式详细描述，将使本发明的技术方案及其他有益效果显而易见。The technical solutions and other beneficial effects of the present invention will be apparent through the detailed description of specific embodiments of the present invention below in conjunction with the accompanying drawings.

实施例1：甘露糖基化的阳离子脂质体复合物Example 1: Mannosylated cationic liposome complexes

本发明提出采用甘露糖基化的阳离子脂质体复合物作为新型疫苗载体，构建安全高效的纳米疫苗。The invention proposes to use the mannosylated cationic liposome complex as a novel vaccine carrier to construct a safe and efficient nano-vaccine.

甘露糖基化的阳离子脂质体复合物的结构参见图1，包括按比例混合的阳离子脂、中性磷脂和甘露糖苷，阳离子脂和中性磷脂的摩尔比为19∶1至2∶18，甘露糖苷与包括阳离子脂和中性磷脂的脂质体的摩尔比为2∶98至20∶80。The structure of the mannosylated cationic liposome complex is shown in Figure 1, including cationic lipids, neutral phospholipids and mannoside mixed in proportion, the molar ratio of cationic lipids and neutral phospholipids is 19:1 to 2:18, The molar ratio of mannoside to liposomes including cationic lipids and neutral phospholipids is 2:98 to 20:80.

阳离子脂是带有正电荷两性分子，包括带有正电荷的头部和疏水尾部，带正电荷的极性头部含有氨基、季铵盐及多胺等胺类基团，而疏水尾部包括饱和或不饱和脂肪酸链或胆固醇环，分子的主链是甘油，甘油的第二个羟基为带正电荷的季铵盐，另两个羟基被饱和或不饱和脂肪酸酯化；阳离子脂包括DDAB、DOTAP、DODAP、DOTMA和DOEPC中的一种或多种的组合。Cationic lipids are positively charged amphoteric molecules, including a positively charged head and a hydrophobic tail. The positively charged polar head contains amine groups such as amino groups, quaternary ammonium salts, and polyamines, while the hydrophobic tail includes saturated Or unsaturated fatty acid chain or cholesterol ring, the main chain of the molecule is glycerol, the second hydroxyl group of glycerol is a positively charged quaternary ammonium salt, and the other two hydroxyl groups are esterified by saturated or unsaturated fatty acids; cationic lipids include DDAB, A combination of one or more of DOTAP, DODAP, DOTMA and DOEPC.

中性磷脂是两性分子，包括磷酸相连的取代基团(含氨碱或醇类)构成的亲水头和脂肪酸链构成的疏水尾，表面静电荷基本接近0，分子的主链是甘油，甘油的第三个羟基被磷酸酯化，另外两个羟基被饱和或不饱和脂肪酸酯化，磷酸基团又与胆碱或胆胺基团相连；中性磷脂包括DOPC、DMPC、DLPC、DPPC、DSPC、DMPE、DPPE和DSPE中的一种或多种的组合。Neutral phospholipids are amphiphilic molecules, including a hydrophilic head composed of phosphoric acid-linked substituent groups (including ammonia bases or alcohols) and a hydrophobic tail composed of fatty acid chains. The surface electrostatic charge is basically close to 0. The main chain of the molecule is glycerol, glycerol The third hydroxyl group is phosphorylated, and the other two hydroxyl groups are esterified by saturated or unsaturated fatty acids, and the phosphoric acid group is connected to choline or cholamine groups; neutral phospholipids include DOPC, DMPC, DLPC, DPPC, A combination of one or more of DSPC, DMPE, DPPE and DSPE.

甘露糖苷包括甲基-D-甘露糖苷、4-硝基苯基-α-D-吡喃甘露糖苷、4-甲基伞形酮基-α-D-吡喃甘露糖苷和4-氨基苯基-D-甘露糖苷中的一种或多种的组合。Mannosides include methyl-D-mannoside, 4-nitrophenyl-α-D-mannopyranoside, 4-methylumbelliferyl-α-D-mannopyranoside, and 4-aminophenyl - a combination of one or more of D-mannosides.

实施例2：具有免疫佐剂功效的疫苗载体Example 2: Vaccine carrier with immune adjuvant effect

本发明的具有免疫佐剂功效的疫苗载体包裹疫苗抗原，疫苗载体为甘露糖基化的阳离子脂质体复合物。The vaccine carrier with immune adjuvant effect of the present invention wraps vaccine antigen, and the vaccine carrier is mannosylated cationic liposome complex.

甘露糖基化的阳离子脂质体复合物的结构参见图1，包括按比例混合的阳离子脂、中性磷脂和甘露糖苷，阳离子脂和中性磷脂的摩尔比为19∶1至2∶18，甘露糖苷与包括阳离子脂和中性磷脂的脂质体的摩尔比为2∶98至20∶80。阳离子脂是带有正电荷两性分子，包括带有正电荷的头部和疏水尾部，带正电荷的极性头部含有氨基、季铵盐及多胺等胺类基团，而疏水尾部包括饱和或不饱和脂肪酸链或胆固醇环，是表面携带正电荷的两性分子，分子的主链是甘油，甘油的第二个羟基为带正电荷的季铵盐，另两个羟基被饱和或不饱和脂肪酸酯化；阳离子脂包括DDAB、DOTAP、DODAP、DOTMA和DOEPC中的一种或多种的组合。中性磷脂是两性分子，包括磷酸相连的取代基团(含氨碱或醇类)构成的亲水头和脂肪酸链构成的疏水尾，表面静电荷基本接近0，分子的主链是甘油，甘油的第三个羟基被磷酸酯化，另外两个羟基被饱和或不饱和脂肪酸酯化，磷酸基团又与胆碱或胆胺基团相连；中性磷脂包括DOPC、DMPC、DLPC、DPPC、DSPC、DMPE、DPPE和DSPE中的一种或多种的组合。甘露糖苷包括甲基-D-甘露糖苷、4-硝基苯基-α-D-吡喃甘露糖苷、4-甲基伞形酮基-α-D-吡喃甘露糖苷和4-氨基苯基-D-甘露糖苷中的一种或多种的组合。The structure of the mannosylated cationic liposome complex is shown in Figure 1, including cationic lipids, neutral phospholipids and mannoside mixed in proportion, the molar ratio of cationic lipids and neutral phospholipids is 19:1 to 2:18, The molar ratio of mannoside to liposomes including cationic lipids and neutral phospholipids is 2:98 to 20:80. Cationic lipids are positively charged amphoteric molecules, including a positively charged head and a hydrophobic tail. The positively charged polar head contains amine groups such as amino groups, quaternary ammonium salts, and polyamines, while the hydrophobic tail includes saturated Or unsaturated fatty acid chain or cholesterol ring, which is an amphipathic molecule with a positive charge on the surface. The main chain of the molecule is glycerol. The second hydroxyl group of glycerol is a positively charged quaternary ammonium salt, and the other two hydroxyl groups are saturated or unsaturated fatty acids. Esterification; cationic lipids include one or more combinations of DDAB, DOTAP, DODAP, DOTMA and DOEPC. Neutral phospholipids are amphiphilic molecules, including a hydrophilic head composed of phosphoric acid-linked substituent groups (including ammonia bases or alcohols) and a hydrophobic tail composed of fatty acid chains. The surface electrostatic charge is basically close to 0. The main chain of the molecule is glycerol, glycerol The third hydroxyl group is phosphorylated, and the other two hydroxyl groups are esterified by saturated or unsaturated fatty acids, and the phosphoric acid group is connected to choline or cholamine groups; neutral phospholipids include DOPC, DMPC, DLPC, DPPC, A combination of one or more of DSPC, DMPE, DPPE and DSPE. Mannosides include methyl-D-mannoside, 4-nitrophenyl-α-D-mannopyranoside, 4-methylumbelliferyl-α-D-mannopyranoside, and 4-aminophenyl - a combination of one or more of D-mannosides.

实施例3：纳米疫苗Embodiment 3: nano vaccine

本发明的纳米疫苗，包括疫苗抗原和包裹疫苗抗原的脂质体，脂质体采用甘露糖基化的阳离子脂质体复合物，抗原为各类蛋白、多肽、多糖、DNA或RNA，抗原来自于病毒、细菌、其他微生物、肿瘤或基因工程蛋白产物。The nano-vaccine of the present invention comprises a vaccine antigen and a liposome encapsulating the vaccine antigen, the liposome adopts mannosylated cationic liposome complex, the antigen is various proteins, polypeptides, polysaccharides, DNA or RNA, and the antigen comes from For viruses, bacteria, other microorganisms, tumors or genetically engineered protein products.

甘露糖基化的阳离子脂质体复合物的结构参见图1，包括按比例混合的阳离子脂、中性磷脂和甘露糖苷，阳离子脂和中性磷脂的摩尔比为19∶1至2∶18，甘露糖苷与包括阳离子脂和中性磷脂的脂质体的摩尔比为2∶98至20∶80。阳离子脂是带有正电荷两性分子，包括带有正电荷的头部和疏水尾部，带正电荷的极性头部含有氨基、季铵盐及多胺等胺类基团，而疏水尾部包括饱和或不饱和脂肪酸链或胆固醇环，是表面携带正电荷的两性分子，分子的主链是甘油，甘油的第二个羟基为带正电荷的季铵盐，另两个羟基被饱和或不饱和脂肪酸酯化；阳离子脂包括DDAB、DOTAP、DODAP、DOTMA和DOEPC中的一种或多种的组合。中性磷脂是两性分子，包括磷酸相连的取代基团(含氨碱或醇类)构成的亲水头和脂肪酸链构成的疏水尾，表面静电荷基本接近0，分子主链是甘油，甘油的第三个羟基被磷酸酯化，另外两个羟基被饱和或不饱和脂肪酸酯化，磷酸基团又与胆碱或胆胺基团相连；中性磷脂包括DOPC、DMPC、DLPC、DPPC、DSPC、DMPE、DPPE和DSPE中的一种或多种的组合。甘露糖苷包括甲基-D-甘露糖苷、4-硝基苯基-α-D-吡喃甘露糖苷、4-甲基伞形酮基-α-D-吡喃甘露糖苷和4-氨基苯基-D-甘露糖苷中的一种或多种的组合。The structure of the mannosylated cationic liposome complex is shown in Figure 1, including cationic lipids, neutral phospholipids and mannoside mixed in proportion, the molar ratio of cationic lipids and neutral phospholipids is 19:1 to 2:18, The molar ratio of mannoside to liposomes including cationic lipids and neutral phospholipids is 2:98 to 20:80. Cationic lipids are positively charged amphoteric molecules, including a positively charged head and a hydrophobic tail. The positively charged polar head contains amine groups such as amino groups, quaternary ammonium salts, and polyamines, while the hydrophobic tail includes saturated Or unsaturated fatty acid chain or cholesterol ring, which is an amphipathic molecule with a positive charge on the surface. The main chain of the molecule is glycerol. The second hydroxyl group of glycerol is a positively charged quaternary ammonium salt, and the other two hydroxyl groups are saturated or unsaturated fatty acids. Esterification; cationic lipids include one or more combinations of DDAB, DOTAP, DODAP, DOTMA and DOEPC. Neutral phospholipids are amphiphilic molecules, including a hydrophilic head composed of phosphoric acid-linked substituent groups (including ammonia bases or alcohols) and a hydrophobic tail composed of fatty acid chains. The surface electrostatic charge is basically close to 0. The main chain of the molecule is glycerol. The third hydroxyl group is phosphorylated, and the other two hydroxyl groups are esterified by saturated or unsaturated fatty acids, and the phosphoric acid group is connected to a choline or cholamine group; neutral phospholipids include DOPC, DMPC, DLPC, DPPC, DSPC A combination of one or more of , DMPE, DPPE and DSPE. Mannosides include methyl-D-mannoside, 4-nitrophenyl-α-D-mannopyranoside, 4-methylumbelliferyl-α-D-mannopyranoside, and 4-aminophenyl - a combination of one or more of D-mannosides.

这种基于阳离子脂质体复合物的疫苗可以采用皮下注射和肌肉注射等方法对机体进行免疫。The vaccine based on the cationic liposome complex can be used to immunize the body by means of subcutaneous injection and intramuscular injection.

实施例4：纳米疫苗的制备方法Embodiment 4: the preparation method of nano vaccine

参见图2，采用具体制备方法如下：Referring to Figure 2, the specific preparation method is as follows:

称取一定量的阳离子脂、中性磷脂和甘露糖苷分别溶于氯仿-甲醇(2∶1)，然后按一定比例混合，置于圆底烧瓶中。用稳定的氮气流旋转吹干，使之成一层均匀的薄膜，而后置于真空干燥箱中真空干燥过夜。第二天加入含有抗原的PBS缓冲液，放置于4℃水化12小时。后经水浴超声10分钟，挤压过聚碳酸酯膜两次，4℃放置备用。A certain amount of cationic lipids, neutral phospholipids and mannosides were weighed and dissolved in chloroform-methanol (2:1), then mixed in a certain proportion and placed in a round bottom flask. Rotate and blow dry with a steady stream of nitrogen to form a uniform film, and then place it in a vacuum oven to dry overnight in vacuum. The next day, PBS buffer containing antigen was added and placed at 4°C for 12 hours of hydration. After that, it was sonicated in a water bath for 10 minutes, squeezed through the polycarbonate film twice, and placed at 4°C for later use.

实验显示，阳离子脂质体复合物可以显著诱导单核细胞表达CD86(一种共刺激分子)，提示该复合物促进单核细胞的活化，而中性脂质体对单核细胞活性无明显影响，参见图3，图中示出本发明的三种实施例，即阳离子脂和中性磷脂的摩尔比分别是19∶1、10∶10和2∶18时的实验数据与对照数据的对比。Experiments showed that the cationic liposome complex could significantly induce the expression of CD86 (a co-stimulatory molecule) in monocytes, suggesting that the complex promotes the activation of monocytes, while neutral liposomes have no significant effect on the activity of monocytes , referring to Fig. 3, three kinds of embodiments of the present invention are shown in the figure, namely the comparison of experimental data and control data when the molar ratio of cationic lipid and neutral phospholipid is 19:1, 10:10 and 2:18 respectively.

阳离子脂质体复合物也显著诱导树突状细胞表达CD83，提示该复合物促进树突状细胞的成熟，参见图4。The cationic liposome complex also significantly induced the expression of CD83 in dendritic cells, suggesting that the complex promotes the maturation of dendritic cells, see Figure 4.

此外，阳离子脂质体复合物为抗原(BSA-FITC)的载体，显著提高了抗原呈递细胞(小鼠巨噬细胞)对抗原的摄取，参见图5，图中示出本发明的三种实施例，即阳离子脂和中性磷脂的摩尔比分别是19∶1、10∶10和2∶18时的实验数据与对照数据的对比。与游离抗原相比，阳离子脂质体复合物提高巨噬细胞对抗原的吞噬率高达10倍以上，这一结果显示阳离子脂质体复合物是一种高效的抗原载体。In addition, the cationic liposome complex is the carrier of the antigen (BSA-FITC), which significantly improves the uptake of the antigen by antigen-presenting cells (mouse macrophages), see Figure 5, which shows three implementations of the present invention For example, the comparison between the experimental data and the control data when the molar ratios of cationic lipids and neutral phospholipids are 19:1, 10:10 and 2:18 respectively. Compared with the free antigen, the cationic liposome complex increased the phagocytosis rate of macrophages to the antigen by more than 10 times. This result shows that the cationic liposome complex is an efficient antigen carrier.

图6的结果显示单纯使用阳离子脂明显降低单核细胞的存活率，提示其具有一定的细胞毒性，图中示出本发明的三种实施例，即阳离子脂和中性磷脂的摩尔比分别是19∶1、10∶10和2∶18时的实验数据与对照数据的对比。而在阳离子脂中掺入中性磷脂制成阳离子脂质体复合物则大大减轻阳离子脂的细胞毒作用，对单核细胞的存活率无明显影响。The result of Fig. 6 shows that using cationic lipids alone significantly reduces the survival rate of monocytes, suggesting that it has certain cytotoxicity. Three kinds of embodiments of the present invention are shown in the figure, that is, the molar ratios of cationic lipids and neutral phospholipids are respectively Comparison of experimental data and control data at 19:1, 10:10 and 2:18. However, neutral phospholipids are mixed into cationic lipids to make cationic liposome complexes, which can greatly reduce the cytotoxic effect of cationic lipids, and have no obvious influence on the survival rate of monocytes.

本发明的甘露糖基化的阳离子脂质体复合物不但可以作为疫苗载体，还可以作为免疫佐剂的载体，提高佐剂的免疫促进作用，减少佐剂带来的副作用。本发明的甘露糖基化的阳离子脂质体复合物不仅可以包裹抗原，还可以联合包裹其他佐剂，以进一步提高疫苗的免疫效果。The mannosylated cationic liposome complex of the present invention can not only be used as a vaccine carrier, but also can be used as a carrier of immune adjuvant to improve the immune promoting effect of the adjuvant and reduce the side effects caused by the adjuvant. The mannosylated cationic liposome complex of the present invention can not only wrap antigens, but also co-wrap other adjuvants to further improve the immune effect of vaccines.

可以理解的是，对本领域普通技术人员来说，可以根据本发明的技术方案及其发明构思加以等同替换或改变，而所有这些改变或替换都应属于本发明所附的权利要求的保护范围。It can be understood that those skilled in the art can make equivalent replacements or changes according to the technical solutions and inventive concepts of the present invention, and all these changes or replacements should belong to the protection scope of the appended claims of the present invention.

Claims (24)

Translated fromChinese

1.一种甘露糖基化的阳离子脂质体复合物，其特征在于，包括按比例混合的阳离子脂、中性磷脂和甘露糖苷，阳离子脂和中性磷脂的摩尔比为19∶1至2∶18，甘露糖苷与包括阳离子脂和中性磷脂的脂质体的摩尔比为2∶98至20∶80。1. A mannosylated cationic liposome complex, characterized in that, comprises cationic lipids, neutral phospholipids and mannosides mixed in proportion, and the mol ratio of cationic lipids and neutral phospholipids is 19: 1 to 2 :18, the molar ratio of mannoside to liposomes including cationic lipids and neutral phospholipids is 2:98 to 20:80.2.根据权利要求1所述的甘露糖基化的阳离子脂质体复合物，其特征在于，所述阳离子脂是带有正电荷两性分子，包括带有正电荷的头部和疏水尾部，带正电荷的极性头部含有氨基、季铵盐及多胺等胺类基团，而疏水尾部包括饱和或不饱和脂肪酸链或胆固醇环。2. the cationic liposome complex of mannosylation according to claim 1, is characterized in that, described cationic fat is to have positively charged amphoteric molecule, comprises the head and hydrophobic tail with positive charge, with The positively charged polar head contains amine groups such as amino groups, quaternary ammonium salts, and polyamines, while the hydrophobic tail includes saturated or unsaturated fatty acid chains or cholesterol rings.3.根据权利要求2所述的甘露糖基化的阳离子脂质体复合物，其特征在于，所述阳离子脂包括双十烷基二甲基溴化铵、二油酰三甲基铵丙烷、二油酰丙基氯化三甲铵、二甲氨基乙基氨甲酰基-胆固醇和二油酰磷脂酰胆碱醚中的一种或多种的组合。3. the cationic liposome complex of mannosylation according to claim 2, is characterized in that, described cationic lipid comprises didecyl dimethyl ammonium bromide, dioleoyl trimethyl ammonium propane, A combination of one or more of dioleoylpropyltrimethylammonium chloride, dimethylaminoethylcarbamoyl-cholesterol and dioleoylphosphatidylcholine ether.4.根据权利要求1至3中任一项所述的甘露糖基化的阳离子脂质体复合物，其特征在于，所述中性磷脂是两性分子，包括磷酸相连的含氨碱或醇类取代基团构成的亲水头和脂肪酸链构成的疏水尾，表面静电荷基本接近0。4. The mannosylated cationic liposome complex according to any one of claims 1 to 3, wherein the neutral phospholipids are amphiphilic molecules comprising phosphoric acid-linked ammoniacal bases or alcohols The hydrophilic head composed of substituent groups and the hydrophobic tail composed of fatty acid chains have a surface electrostatic charge close to zero.5.根据权利要求4所述的甘露糖基化的阳离子脂质体复合物，其特征在于，所述中性磷脂包括二油酰磷脂酰胆碱、二豆蔻酰磷脂酰胆碱、二亚油酰磷脂酰胆碱、二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二肉豆蔻酰磷酸乙醇胺、二棕榈酰磷脂酰乙醇胺和二硬脂酰磷脂酰乙醇胺中的一种或多种的组合。5. the cationic liposome complex of mannosylation according to claim 4, is characterized in that, described neutral phospholipid comprises dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dilinoleic acid One or more of acylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine and distearoylphosphatidylethanolamine combination of species.6.根据权利要求4所述的甘露糖基化的阳离子脂质体复合物，其特征在于，所述甘露糖苷包括甲基-D-甘露糖苷、4-硝基苯基-α-D-吡喃甘露糖苷、4-甲基伞形酮基-α-D-吡喃甘露糖苷和4-氨基苯基-D-甘露糖苷中的一种或多种的组合。6. The cationic liposome complex of mannosylation according to claim 4, wherein said mannoside comprises methyl-D-mannoside, 4-nitrophenyl-α-D-pyrroside A combination of one or more of mannoside, 4-methylumbelliferyl-α-D-mannopyranoside, and 4-aminophenyl-D-mannoside.7.一种具有免疫佐剂功效的疫苗载体，包裹疫苗抗原，其特征在于，所述疫苗载体为甘露糖基化的阳离子脂质体复合物，包括按比例混合的阳离子脂、中性磷脂和甘露糖苷，阳离子脂和中性磷脂的摩尔比为19∶1至2∶18，甘露糖苷与包括阳离子脂和中性磷脂的脂质体的摩尔比为2∶98至20∶80。7. A vaccine carrier with immune adjuvant effect, wrapped vaccine antigen, is characterized in that, described vaccine carrier is the cationic liposome complex of mannosylation, comprises the cationic fat that mixes in proportion, neutral phospholipid and The molar ratio of mannoside, cationic lipid and neutral phospholipid is 19:1 to 2:18, and the molar ratio of mannoside to liposome including cationic lipid and neutral phospholipid is 2:98 to 20:80.8.根据权利要求7所述的具有免疫佐剂功效的疫苗载体，其特征在于，所述阳离子脂是带有正电荷两性分子，包括带有正电荷的头部和疏水尾部，带正电荷的极性头部含有氨基、季铵盐及多胺等胺类基团，而疏水尾部包括饱和或不饱和脂肪酸链或胆固醇环。8. The vaccine carrier with immune adjuvant effect according to claim 7, characterized in that, the cationic lipid is a positively charged amphiphilic molecule, comprising a positively charged head and a hydrophobic tail, and a positively charged The polar head contains amine groups such as amino groups, quaternary ammonium salts, and polyamines, while the hydrophobic tail includes saturated or unsaturated fatty acid chains or cholesterol rings.9.根据权利要求8所述的具有免疫佐剂功效的疫苗载体，其特征在于，所 述阳离子脂包括双十烷基二甲基溴化铵、二油酰三甲基铵丙烷、二油酰丙基氯化三甲铵、二甲氨基乙基氨甲酰基-胆固醇和二油酰磷脂酰胆碱醚中的一种或多种的组合。9. the vaccine carrier with immune adjuvant effect according to claim 8, is characterized in that, described cationic fat comprises didecyl dimethyl ammonium bromide, dioleoyl trimethyl ammonium propane, dioleoyl A combination of one or more of propyltrimethylammonium chloride, dimethylaminoethylcarbamoyl-cholesterol and dioleoylphosphatidylcholine ether.10.根据权利要求7至9中任一项所述的具有免疫佐剂功效的疫苗载体，其特征在于，所述中性磷脂是两性分子，包括磷酸相连的含氨碱或醇类取代基团构成的亲水头和脂肪酸链构成的疏水尾，表面静电荷基本接近0。10. The vaccine carrier with immune adjuvant effect according to any one of claims 7 to 9, characterized in that, the neutral phospholipid is an amphiphilic molecule, comprising a phosphoric acid-linked ammonia-containing base or an alcohol substituent group The hydrophilic head and the hydrophobic tail composed of fatty acid chains have a surface electrostatic charge close to zero.11.根据权利要求10所述的具有免疫佐剂功效的疫苗载体，其特征在于，所述中性磷脂包括二油酰磷脂酰胆碱、二豆蔻酰磷脂酰胆碱、二亚油酰磷脂酰胆碱、二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二肉豆蔻酰磷酸乙醇胺、二棕榈酰磷脂酰乙醇胺和二硬脂酰磷脂酰乙醇胺中的一种或多种的组合。11. The vaccine carrier with immune adjuvant effect according to claim 10, characterized in that, said neutral phospholipids include dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dilinoleoylphosphatidylcholine Combination of one or more of choline, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine and distearoylphosphatidylethanolamine .12.根据权利要求10所述的具有免疫佐剂功效的疫苗载体，其特征在于，所述甘露糖苷包括甲基-D-甘露糖苷、4-硝基苯基-α-D-吡喃甘露糖苷、4-甲基伞形酮基-α-D-吡喃甘露糖苷和4-氨基苯基-D-甘露糖苷中的一种或多种的组合。12. The vaccine carrier with immune adjuvant effect according to claim 10, characterized in that, the mannoside comprises methyl-D-mannoside, 4-nitrophenyl-α-D-mannopyranoside A combination of one or more of , 4-methylumbelliferyl-α-D-mannopyranoside and 4-aminophenyl-D-mannoside.13.一种纳米疫苗，包括疫苗抗原和包裹疫苗抗原的脂质体，其特征在于，所述脂质体采用甘露糖基化的阳离子脂质体复合物，包括按比例混合的阳离子脂、中性磷脂和甘露糖苷，阳离子脂和中性磷脂的摩尔比为19∶1至2∶18，甘露糖苷与包括阳离子脂和中性磷脂的脂质体的摩尔比为2∶98至20∶80。13. A nano-vaccine, comprising a vaccine antigen and a liposome encapsulating the vaccine antigen, characterized in that the liposome adopts a mannosylated cationic liposome complex, comprising cationic lipids mixed in proportion, medium The molar ratio of phospholipids and mannosides, cationic lipids and neutral phospholipids is 19:1 to 2:18, and the molar ratio of mannoside to liposomes including cationic lipids and neutral phospholipids is 2:98 to 20:80.14.根据权利要求13所述的纳米疫苗，其特征在于，所述阳离子脂是带有正电荷两性分子，包括带有正电荷的头部和疏水尾部，带正电荷的极性头部含有氨基、季铵盐及多胺等胺类基团，而疏水尾部包括饱和或不饱和脂肪酸链或胆固醇环。14. The nano-vaccine according to claim 13, wherein the cationic lipid is a positively charged amphiphilic molecule, comprising a positively charged head and a hydrophobic tail, and the positively charged polar head contains an amino group , quaternary ammonium salts and polyamines and other amine groups, while the hydrophobic tail includes saturated or unsaturated fatty acid chains or cholesterol rings.15.根据权利要求14所述的纳米疫苗，其特征在于，所述阳离阳离子脂包括双十烷基二甲基溴化铵、二油酰三甲基铵丙烷、二油酰丙基氯化三甲铵、二甲氨基乙基氨甲酰基-胆固醇和二油酰磷脂酰胆碱醚中的一种或多种的组合。15. nano vaccine according to claim 14, is characterized in that, described cationic cationic lipid comprises didecyl dimethyl ammonium bromide, dioleoyl trimethyl ammonium propane, dioleoyl propyl chloride A combination of one or more of trimethylammonium, dimethylaminoethylcarbamoyl-cholesterol, and dioleoylphosphatidylcholine ether.16.根据权利要求13至15中任一项所述的纳米疫苗，其特征在于，所述中性磷脂是两性分子，包括磷酸相连的含氨碱或醇类取代基团构成的亲水头和脂肪酸链构成的疏水尾，表面静电荷基本接近0。16. The nano-vaccine according to any one of claims 13 to 15, wherein the neutral phospholipid is an amphiphilic molecule, comprising a hydrophilic head composed of a phosphoric acid-linked ammonia-containing base or an alcohol substituent group and The hydrophobic tail composed of fatty acid chains has a surface electrostatic charge close to zero.17.根据权利要求16所述的纳米疫苗，其特征在于，所述中性磷脂包括二油酰磷脂酰胆碱、二豆蔻酰磷脂酰胆碱、二亚油酰磷脂酰胆碱、二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二肉豆蔻酰磷酸乙醇胺、二棕榈酰磷脂酰乙醇 胺和二硬脂酰磷脂酰乙醇胺中的一种或多种的组合。17. The nano vaccine according to claim 16, wherein the neutral phospholipids include dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dilinoleoylphosphatidylcholine, dipalmitoyl A combination of one or more of phosphatidylcholine, distearoylphosphatidylcholine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine and distearoylphosphatidylethanolamine.18.根据权利要求16所述的纳米疫苗，其特征在于，所述甘露糖苷包括甲基-D-甘露糖苷、4-硝基苯基-α-D-吡喃甘露糖苷、4-甲基伞形酮基-α-D-吡喃甘露糖苷和4-氨基苯基-D-甘露糖苷中的一种或多种的组合。18. The nano-vaccine according to claim 16, wherein the mannosides include methyl-D-mannoside, 4-nitrophenyl-α-D-mannopyranoside, 4-methylumbrella A combination of one or more of keto-α-D-mannopyranoside and 4-aminophenyl-D-mannoside.19.根据权利要求16所述的纳米疫苗，其特征在于，所述抗原为各类蛋白、多肽、多糖、DNA或RNA，所述抗原来自于病毒、细菌、其他微生物、肿瘤或基因工程蛋白产物。19. The nano-vaccine according to claim 16, characterized in that, the antigens are various proteins, polypeptides, polysaccharides, DNA or RNA, and the antigens are derived from viruses, bacteria, other microorganisms, tumors or genetically engineered protein products .20.根据权利要求13所述的纳米疫苗的制备方法，包括如下步骤：20. the preparation method of nano vaccine according to claim 13, comprises the steps:取阳离子脂、中性磷脂和甘露糖苷分别溶于氯仿-甲醇，然后按比例混合后置于容器中；Dissolve cationic lipids, neutral phospholipids and mannosides in chloroform-methanol respectively, then mix them in proportion and place them in a container;将容器中的混合物吹干成一层均匀的薄膜，而后进行真空干燥处理；Dry the mixture in the container to form a uniform film, and then carry out vacuum drying;加入含有抗原的缓冲液，然后进行水化处理及水浴超声处理，挤压过聚碳酸酯膜，放置备用。Add the buffer solution containing the antigen, then carry out hydration treatment and water bath ultrasonic treatment, extrude the polycarbonate membrane, and put it aside for later use.21.根据权利要求20所述的方法，其特征在于，所述氯仿-甲醇中氯仿和甲醇的体积比为2∶1。21. The method according to claim 20, characterized in that, the volume ratio of chloroform and methanol in the chloroform-methanol is 2:1.22.根据权利要求20的方法，其特征在于，所述容器中的混合物用稳定的氮气流旋转吹干。22. The method according to claim 20, characterized in that the mixture in the container is rotary blown dry with a steady stream of nitrogen.23.根据权利要求20的方法，其特征在于，真空干燥处理是在真空干燥箱中真空干燥过夜，第二天加入含有抗原的PBS缓冲液。23. The method according to claim 20, characterized in that the vacuum drying treatment is vacuum drying overnight in a vacuum drying oven, and adding PBS buffer solution containing the antigen the next day.24.根据权利要求20的方法，其特征在于，加入含有抗原的磷酸盐缓冲液后，放置于4℃水化12小时，水浴超声10分钟，挤压过聚碳酸酯膜两次，4℃放置备用。 24. The method according to claim 20, characterized in that, after adding the phosphate buffer containing the antigen, place it at 4°C for hydration for 12 hours, ultrasonicate it in a water bath for 10 minutes, squeeze the polycarbonate membrane twice, place it at 4°C spare. the

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